PROJECT SUMMARY Sepsis, commonly caused by bloodstream infections (BSI), is a rapidly progressive and life-threatening disease. Unfortunately, prolonged delay in microbiological diagnosis increases patient mortality, promotes the misuse of antibiotics, and consequently, the evolution of antibiotic-resistant pathogens. Herein, we aim to deliver an amplification-free, microfluidic system for pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) directly from whole blood. To achieve our goal, we propose a platform based on microfluidic- assisted microscopy to sort, trap, detect, and monitor pathogens at single cell resolution. For pathogen ID, we will adopt a multispectral barcoding scheme to differentially label molecular probes for direct multiplex ribosomal RNA (rRNA) detection to classify and speciate pathogens, along with a nanotube assisted microwave electroporation (NAME) technique to efficiently deliver the probes intracellularly for amplification-free single microbe detection. Positive pathogen ID will guide quantitative multimodal phenotypic AST (mPhAST), in which we will monitor early changes in microbial growth kinetics with cytological measures of viability in response to relevant antibiotic conditions at the single cell level to determine susceptibility/resistance with improved speed and reliability. Combined with upstream whole blood pre-processing for pathogen isolation and concentration followed by ID then AST, we aim to deliver sample to answer within 90 min for BSI triage and as early as 30 minutes more for antibiotic minimum inhibitory concentration (MIC) determination. We have assembled a superb team of multi-disciplinary investigators and industry-leading advisors with complementary expertise and a strong track record of collaboration. We propose the following aims: 1) to develop a rapid BSI triage protocol for broad pathogen detection, classification, and ID; 2) to develop a quantitative mPhAST; 3) to develop an integrated ID- mPhAST platform; 4) to perform analytical and clinical validation of our ID-mPhAST platform. Our short-term goal is to obtain the necessary preliminary data to plan for product development and commercialization, with the long-term goal of translating our diagnostic platform to reduce sepsis-related morbidity and mortality.